title: Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function.
abstract: Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.
authors.affiliation: Division of Immunology, Hospital General Universitario Gregorio Marañón, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Ramon
authors.initials: R
authors.lastname: Urrea Moreno
authors.forename: Juana
authors.initials: J
authors.lastname: Gil
authors.forename: Carmen
authors.initials: C
authors.lastname: Rodriguez-Sainz
authors.forename: Elena
authors.initials: E
authors.lastname: Cela
authors.forename: Victor
authors.initials: V
authors.lastname: LaFay
authors.forename: Brian
authors.initials: B
authors.lastname: Oloizia
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Herr
authors.forename: Janos
authors.initials: J
authors.lastname: Sumegi
authors.forename: Michael B
authors.initials: MB
authors.lastname: Jordan
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Risma
mesh_terms: D019943:Amino Acid Substitution / Q000276:immunology; D000818:Animals; D002118:Calcium / Q000276:immunology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology*; D006706:Homeostasis / Q000276:immunology; D006801:Humans; D007111:Immunity, Cellular / Q000235:genetics / Q000276:immunology; D008563:Membrane Lipids / Q000276:immunology*; D051379:Mice; D020125:Mutation, Missense / Q000276:immunology*; D054353:Perforin / Q000235:genetics / Q000276:immunology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D008563:Membrane Lipids; D054353:Perforin; D002118:Calcium